Clinical heterogeneity and a high proportion of novel mutations in a Chinese cohort of patients with dysferlinopathy

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1 Original Article Clinical heterogeneity and a high proportion of novel mutations in a Chinese cohort of patients with dysferlinopathy Jianying Xi 1,2, Gaëlle Blandin 3,4, Jiahong Lu 1,2, Sushan Luo 1,2, Wenhua Zhu 1,2, Christophe Béroud 3,4,5, Christophe Pécheux 5, Véronique Labelle 5, Nicolas Lévy 3,4,5, Jon Andoni Urtizberea 6, Chongbo Zhao 1,2, Martin Krahn 3,4,5 1 Department of Neurology, Huashan Hospital, Fudan University, , Shanghai, China, 2 Fudan University, Institute of Neurology, , Shanghai, China, 3 Aix Marseille University, GMGF, 13385, Marseille, France, 4 Inserm, UMR_S 910, 13385, Marseille, France, 5 Department of Medical Genetics and Cell Biology, APHM, Children s Hospital La Timone, 13385, Marseille, France, 6 APHP, Marin Hospital, 64700, Hendaye, France Abstract Address for correspondence: Dr. Martin Krahn, Université d Aix Marseille, Faculté de Médecine de Marseille, 4e étage Aile Verte, 27 boulevard Jean Moulin, Marseille Cedex 05, France. E mail: martin.krahn@univ amu.fr Received : Review completed : Accepted : Background and Aims: Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene. This study presents clinical features and the mutational spectrum in the largest cohort of Chinese patients analyzed to date. Patients and Methods: A total of 36 unrelated Chinese patients with diagnostic suspicion of dysferlinopathy were clinically and genetically characterized. Results: Patients were divided into five phenotypes: 19 patients with limb girdle muscular dystrophy (LGMD) type 2B, 10 with Miyoshi myopathy (MM), 1 with distal anterior compartment myopathy (DACM), 3 with exercise intolerance, and 3 with asymptomatic hypercreatine phosphokinasemia (hypercpkemia). Thirty one patients showed an absence or drastic reduction of dysferlin expression by Westernblot. Forty three mutations were identified in DYSF, including 31 novel. Conclusion: Our study underlines clinical heterogeneity and a high proportion of novel mutations in Chinese patients affected with dysferlinopathy. Key words: Dysferlin, distal anterior compartment myopathy, limb girdle muscular dystrophy, Miyoshi myopathy, Mutation, Western blot Introduction Mutations in DYSF (MIM#603009) cause dysferlinopathies, mainly presenting as limb girdle muscular dystrophy type 2B (LGMD2B; MIM#253601) [1] and Miyoshi myopathy (MM; MIM#254130). [2,3] Dysferlinopathies have been reported in different populations, [4] including 15 Chinese patients with genetic studies. [5 7] We report mutational data from the largest Chinese cohort to date, Quick Response Code: Access this article online Neurology India Nov-Dec 2014 Vol 62 Issue 6 Website: PMID: *** DOI: / including 31 novel disease causing mutations which point to a specific mutational spectrum. Patients and Methods After approval by the Huashan Hospital (Fudan University) Institutional Review Board, 36 index patients (admitted to Huashan Hospital during ) from the Han population were enrolled after informed consent, including one patient (C ) previously reported. [6] Dysferlin immunolabeling was negative in 30 patients and reduced in 4 by immunochemistry. Two additional patients without muscle biopsy were included based on clinical symptoms. Detailed clinical information was collected prospectively, including muscle strength (Medical Research Council scale) and functional status (Gardner Medwin and Walton scales. [8]. Single dysferlin Westernblot was 635

2 Table 1: Clinical, pathological, and protein and mutational data obtained in the cohort UMD DYSF identifier Sex Age at onset Age at biopsy Consanguinity (family history) Phenotype Scapular winging Standing on tiptoe Walton scales Serum CPK (U/L) Dysferlin immunochemistry Muscle Western blot dysferlin Calpain 3 (~90 kd) Calpain 3 (~60 kd) Mutational data: Localisation, nomenclature on cdna (and protein), zygosity 2 mut patients F Female LGMD No No 5/M 10,837 ( ) ( ) ( ) (+) Exon24 c. 2490_2509del* (p.leu831ilefsx27) HOZ F Female LGMD No No 7/R 5012 ( ) ( ) (+) (+) Exon30 c. 3220_3221delCT* (p.leu1074phefsx39) HOZ F Female LGMD, myositis like No No 5/R >10,000 ( ) ( ) ( ) 40% Exon23 c. 2204_2205delAC* (p.his735profsx18) HOZ F Male MM No No 2/S 5934 ( ) ( ) (+) (+) Exon33 c. 3601C>T* (p.gln1201x) HOZ F Male MM No No 3/S 3829 ( ) ( ) 49% (+) Exon19 c. 1667T>C* (p.leu556pro) HOZ F Female DACM No Yes 5/M 4545 ( ) ( ) ND ND Exon5 c. 347_350dupCGCT* (p.val118alafsx31) HTZ F Male LGMD No No 8/R 7889 ( ) ( ) ( ) (+) Exon9 c. 863A>T* (p.asp288val) HTZ Exon29 c. 3137G>A (p.arg1046his) HTZ F Male brother LGMD No No 5/M 8541 ( ) ( ) ( ) (+) Exon2 c. 140_143dupATGA* (p.gly49x) HTZ Intron44 c T>C* HTZ F Female LGMD No No 2/S 4950 ( ) ( ) ( ) (+) Exon6 c. 482_483delAG* (p.glu161glyfsx8) HTZ Exon11 c. 965T>C* (p.leu322pro) HTZ F Male LGMD Bilateral No 6/M 8834 ( ) ( ) 80% (+) Exon15 c. 1375dupA* (p.met459asnfsx15) HTZ Exon38 c. 4062_4063insT* (p.pro1355serfsx27) HTZ F Female LGMD No No 3/S 6871 ( ) ( ) 71% (+) Exon20 c. 1759delC* (p.arg588glyfsx39) HTZ Intron45 c G>A* HTZ F Male LGMD No No 8/M 3000 ( ) ( ) (+) (+) Exon2 c. 136T>C* (p.trp46arg) HTZ Exon50 c. 5570A>C* (p.his1857pro) HTZ F Male LGMD No No 5/M 6086 ( ) ( ) (+) (+) Exon23 c. 2193delC* (p.ser732leufsx17) HTZ Exon33 c. 3601C>T* (p.gln12101x) HTZ F Female LGMD No No 4/M 9860 ( ) ( ) ( ) 79% Exon29 c. 3112C>T (p.arg1038x) HTZ Exon46 c. 5098delC* (p.gln1700serfsx22) HTZ F Male LGMD, myositis like No No 5/R Reduced ( ) 24% (+) Exon28 c. 2940delG* (p.leu981phefsx76) HTZ Exon30 c. 3220_3221delCT* (p.leu1074phefsx39) HTZ F Male LGMD No No 5/R ( ) ( ) ( ) (+) Exon39 c. 4325delG* (p.gly1442valfsx6) HTZ Exon19 c. 1667T>C* (p.leu556pro) HTZ C Male sisters MM No No 5/M 5000 ND ND ND ND Intron10 c G>A HTZ Exon13 c. 1277G>T (p.gly426val) HTZ 636 Neurology India Nov-Dec 2014 Vol 62 Issue 6 Contd...

3 Table 1: Contd... UMD DYSF identifier Sex Age at onset Age at biopsy Consanguinity (family history) Phenotype Scapular winging Standing on tiptoe Walton scales Serum CPK (U/L) Dysferlin immunochemistry Muscle Western blot dysferlin Calpain 3 (~90 kd) Calpain 3 (~60 kd) Mutational data: Localisation, nomenclature on cdna (and protein), zygosity F Female brother LGMD No No 7/M 2000 ND ND ND ND Exon8 c. 799_800del (p.phe267leufsx5) HTZ Exon24 c. 2360A>G* (p.gln787arg) HTZ F Female MM No No 3/S 5682 ( ) ( ) ND ND Exon4 c. 329delA* (p.lys110serfsx41) HTZ Exon47 c. 5302C>T (p.arg1768trp) HTZ F Male MM No No 2/S 7330 Reduced 37% 40% (+) Intron50 c G>A HTZ Exon54 c. 6124C>T (p.arg2042cys) HTZ F Male MM No No 2/S ( ) ( ) 17% 70% Exon23 c. 2204_2205delAC* (p.his735profsx18) HTZ Exon43 c. 4739T>A* (p.val1580asp) HTZ F Female MM No No 3/S 3357 ( ) ND ND ND Exon19 c. 1663C>T (p.arg555trp) HTZ Exon30 c. 3220_3221delCT* (p.leu1074phefsx39) HTZ F Male MM No No 3/S 6425 ( ) ( ) ( ) (+) Exon15 c. 1375dupA* (p.met459asnfsx15) HTZ Exon49 c. 5461A>T* (p.arg1821x) HTZ F Male Exercise No Yes 2/S >30000 ( ) ( ) (+) (+) Exon29 c. 3113G>A (p.arg1038gln) HTZ Exon37 c. 3988C>T* (p.gln1330x) HTZ F Female Exercise intolerance No Yes 1/S ( ) ( ) 58% (+) Intron13 c T>C HTZ Exon45 c. 5032T>C* (p.cys1678arg) HTZ F Male Asymptomatic hypercpkaemia No Yes 1/S 8375 ( ) ( ) (+) (+) Exon9 c. 863A>T* (p.asp288val) HTZ Exon11 c. 965T>C* (p.leu322pro) HTZ 1/0 mut patients F Female LGMD No No 6/M 2754 ( ) 5% 58% (+) Exon11 c. 965T>C* (p.leu322pro) HTZ Exon29 c. 3094A>G*, (p.lys1032glu) HTZ F Male LGMD Bilateral No 5/M 4276 ( ) ND ND ND Exon32 c. 3516_3517delTT (p.ser1173x) HTZ Exon33 c. 3618C>T*, (p.tyr1206tyr) HTZ F Male Exercise intolerance F Male Asymptomatic hyperckaemia F Female Grandmother Asymptomatic hypercpkaemia No No 3/S >10,000 ( ) ( ) 4% 60% Intron10 c G>A* HTZ Exon29 c. 3094A>G*, (p.lys1032glu) HTZ No No 1/S ( ) 11% 39% 62% Exon29 c. 3116G>C* (p.arg1039pro) HTZ Intron 51 c C>T*, HTZ No Yes 1/S >30000 ( ) ( ) ( ) (+) Exon29 c. 3113G>A (p.arg1038gln) HTZ Intron20 c C >T*, HTZ Contd... Neurology India Nov-Dec 2014 Vol 62 Issue 6 637

4 Table 1: Contd... UMD DYSF identifier Sex Age at onset Age at biopsy Consanguinity (family history) Phenotype Scapular winging Standing on tiptoe Walton scales Serum CPK (U/L) Dysferlin immunochemistry Muscle Western blot dysferlin Calpain 3 (~90 kd) Calpain 3 (~60 kd) Mutational data: Localisation, nomenclature on cdna (and protein), zygosity F Male MM No No 2/S ( ) ( ) ND ND Exon35 c. 3863delC* (p.pro1288leufsx57) HTZ F Female MM No No 2/S 8567 ( ) ( ) 22% (+) Exon8 c. 799_800delTT (p.phe267leufsx5) HTZ F Female LGMD, myositis like No No 8/R 3000 Reduced ( ) ( ) 65% Exon29 c. 3064C>T* (p.arg1022trp) HTZ 403 Female LGMD No No 4/M Reduced ( ) ( ) (+) No mutation found 416 Male LGMD No No 4/M 4450 ( ) ND ND ND No mutation found 2/1/0 mut=patients with 2, 1, or no clearly disease causing mutations, respectively. CPK Creatine phosphokinase, ND Not done, HOZ Homozygous, HTZ Heterozygous, MM Miyoshi myopathy, LGMD Limb girdle muscular dystrophy, DACM Distal anterior compartment myopathy. *Novel mutation, Mutation is predicted as nonpathogenic using UMD predictor, Bioinformatics analyses predict no deleterious effect of the mutation, Gardner-Medwin and Walton scales: grade 0 subclinical, hyperckemia, and tolerance of normal activities, grade 1 normal gait, unable to run freely, grade 2 abnormal gait, grade 3 muscle weakness, climbing stairs with support, grade 4 positive Gower sign, grade 5 unable to rise from floor, grade 6 unable to climb stairs, grade 7 unable to get up from chairs, grade 8 unable to walk independently, grade 9 unable to eat, drink, or sit without support, Disease progression was evaluated using three grades: S slow progression, only 1 grade change during the past 5 years, M moderate progression, 2 grade changes during the past 5 years R rapid progression, 3 or more grade changes during the past 5 years performed in three patients and multi Westernblot. [9] (dysferlin and calpain 3) in 28 patients. Genomic DYSF mutation screening was conducted as described. [4,10] Results All patients had normal motor milestones. Average age of onset was 23.7 ± 7.7 years (range years) [Table 1]. The sex ratio was 4:5 (16 females versus 20 males). Nineteen patients (52.8%) presented as LGMD, 10 (27.8%) as MM, 1 (2.8%) as distal anterior compartment myopathy (DACM), 3 (8.3%) as exercise intolerance, and 3 (8.3%) as asymptomatic hypercreatine phosphokinasemia (hypercpkemia). In this cohort weakness of the lower extremities occurred earlier and became more severe than that of the upper extremities. Two patients lost ambulation by age 40 and 47. No facial, pharyngeal, and respiratory muscles weakness and no abnormal electrocardiogram or echocardiogram was documented. All 36 patients had very high serum CPK levels (10 150N). Electromyography (EMG) showed a myopathic pattern in all patients. The ~ 230kDa dysferlin band was absent in 28/31 (90.3%) patients and markedly reduced in 3/31 (9.7%). Absence or reduction of the ~90 kda calpain 3 band was evidenced in 22/28 (78.6%) patients (11 absence and 11 reduction). However, only 6/28 (21%) showed secondary reduction for the ~60 kda band [Table 1]. Mutational analyses evidenced 43 different disease causing mutations (including 31 not previously reported) in 34 patients, including [Table 1]: 17 missense (11 novel; pathogenic effect predicted using UMD predictor), 4 nonsense (3 novel), 17 exonic frame shifting mutations (insertions or deletions, 15 novel), and 5 intronic mutations (2 novel; pathogenic effect predicted using UMD Predictor). Twenty one exonic mutations are predicted to disrupt the open reading frame and/or to lead to a premature stop codon. Five variants are predicted to promote abnormal splicing causing a frame shift at the messenger level, either by disrupting a canonical 5 splice donor site (c G > A, c T > C, c T > C, c G > A) or by creating a novel 3 splice acceptor site (c G > A). In total, 58 different disease causing alleles were identified, with 48 and 5 at the heterozygous and homozygous state, respectively. Molecular diagnosis of dysferlinopathy was confirmed in 26/36 (72.2%) patients, including 21 compound heterozygotes and 5 homozygotes (4 with known consanguinity). Based on protein analysis, the diagnosis of dysferlinopathy in the other 10 patients was retained, but could not yet be confirmed at the genetics level. Discussion Dysferlinopathy is the second most frequent type of LGMD in Europe and Japan, [11,12] but is under diagnosed in China. Here we present the data from the largest Chinese cohort characterized to date. Patients with LGMD2B, MM, and DACM initially had weakness of lower extremities, either with proximal or distal involvement; but distal weakness, especially progressive loss of the ability to stand on tiptoe was an important clue leading to the diagnosis. Atypical patients who showed 638 Neurology India Nov-Dec 2014 Vol 62 Issue 6

5 exercise intolerance or asymptomatic hyper CPKemia without muscle weakness were reported before, [3,13,14] and they may be misdiagnosed as metabolic myopathy. However, the CPK level was constantly very high in patients with dysferlinopathies, while it was fluctuating in patients with metabolic myopathies. Thus, various phenotypes in our cohort further emphasized the clinical heterogeneity in Chinese patients with dysferlinopathies. Mutational data analysis in this cohort evidenced a large mutational spectrum. Interestingly, most of the identified disease causing mutations (31, 72%) had not been reported to date. Comparison of the mutational spectrum to a large French cohort [10] points to a possible difference, with a lower proportion of null mutations in the Chinese cohort (62 vs 76%) and accordingly more missense mutations (38 vs 24%). When compared only to patients from Japan (56 index cases in UMD DYSF v1.1 (www. umd.be/dysf/)), no difference in the proportion of mutation types were observed. Two mutations were recurrent (c. 965T > C and c. 3220_3221delCT, both identified in three unrelated patients). Two mutations previously identified in Chinese patients were retrieved in our cohort: c G > A 6 and c. 3112C > T. 7 Altogether, our study underlines the clinical heterogeneity and reports a high proportion of novel mutations in Chinese patients with dysferlinopathy, outlining the importance of further characterization of this disease in China. Acknowledgments We sincerely thank the patients for their participation. We sincerely thank the Association Française contre les Myopathies, the Jain Foundation, the APHM, Inserm, and Aix Marseille Université for funding this work. References 1. Bashir R, Britton S, Strachan T, Keers S, Vafiadaki E, Lako M, et al. A gene related to Caenorhabditis elegans spermatogenesis factor fer 1 is mutated in limb girdle muscular dystrophy type 2B. Nat Genet 1998;20: Liu J, Aoki M, Illa I, Wu C, Fardeau M, Angelini C, et al. Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 1998;20: Nguyen K, Bassez G, Krahn M, Bernard R, Laforêt P, Labelle V, et al. Phenotypic study in 40 patients with dysferlin gene mutations: High frequency of atypical phenotypes. Arch Neurol 2007;64: Blandin G, Beroud C, Labelle V, Nguyen K, Wein N, Hamroun D, et al. UMD DYSF, a novel locus specific database for the compilation and interactive analysis of mutations in the dysferlin gene. Hum Mutat 2012;33:E Shunchang S, Fan Q, Huacheng W, Leturcq F, Yongjian S, Bingfeng Z, et al. Dysferlin mutation in a Chinese pedigree with Miyoshi myopathy. Clin Neurol Neurosurg 2006;108: Ro LS, Lee Chen GJ, Lin TC, Wu YR, Chen CM, Lin CY, et al. Phenotypic features and genetic findings in 2 Chinese families with Miyoshi distal myopathy. Arch Neurol 2004;61: Zhao Z, Hu J, Sakiyama Y, Okamoto Y, Higuchi I, Li N, et al. DYSF mutation analysis in a group of Chinese patients with dysferlinopathy. Clin Neurol Neurosurg 2003;115: Gardner Medwin D, Walton JN. The clinical examination of the voluntary muscles. In: Walton JN, editor. Disorders of the Voluntary Muscles. 3 rd ed. Edinburg: Churchill Livingstone; p Luo SS, Xi JY, Lu JH, Zhao CB, Zhu WH, Lin J, et al. Clinical and pathological features in 15 Chinese patients with calpainopathy. Muscle Nerve 2011;43: Krahn M, Beroud C, Labelle V, Nguyen K, Bernard R, Bassez G, et al. Analysis of the DYSF mutational spectrum in a large cohort of patients. Hum Mutat 2009;30:E Guglieri M, Magri F, D Angelo MG, Prelle A, Morandi L, Rodolico C, et al. Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients. Hum Mutat 2008;29: Tagawa K, Ogawa M, Kawabe K, Yamanaka G, Matsumura T, Goto K, et al. Protein and gene analyses of dysferlinopathy in a large group of Japanese muscular dystrophy patients. J Neurol Sci 2003;211: Nguyen K, Bassez G, Bernard R, Krahn M, Labelle V, Figarella Branger D, et al. Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies. Hum Mutat 2005;26: Okahashi S, Ogawa G, Suzuki M, Ogata K, Nishino I, Kawai M. Asymptomatic sporadic dysferlinopathy presenting with elevation of serum creatine kinase. Typical distribution of muscle involvement shown by MRI but not by CT. Intern Med 2008;47: How to cite this article: Xi J, Blandin G, Lu J, Luo S, Zhu W, Beroud C, et al. Clinical heterogeneity and a high proportion of novel mutations in a Chinese cohort of patients with dysferlinopathy. Neurol India 2014;62: Source of Support: Nil, Conflict of Interest: None declared. Neurology India Nov-Dec 2014 Vol 62 Issue 6 639

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